effect of micronutrients foliar application and ... · they also play a role indirectly through the...

702

Journal of Soil Science and Plant Nutrition, 2016, 16 (3) 2016, 702-715

REEARCH ARTICLE

Effect of micronutrients foliar application and biofertilizeres on essential oils of lemon balm

M. Yadegari*

Department of Agronomy and Medicinal Plants, Faculty of Agriculture, Shahrekord Branch, Islamic Azad University, Po. Box:166. Shahrekord, Iran. *Corresponding author: [email protected]

Abstract

In this work, the effects of two micronutrients (Mn2+, H2Bo3-) and macronutrients (PO43- and NO3

-) from biofer-tilizeres resources on growth (leaf dry/fresh weight, root dry/fresh weight, stem/root length, number of stem) and accumulation of essential oils in Lemon balm (Melissa officinalis L.) were investigated. The most of es-sential oils in two seasons were Caryophyllene oxide, E-Caryophyllene, Geranial, Geraniol, Chavicol and Neral that affected by the treatments. Of the two micronutrients, manganese, and in macronutrients NO3

- were more effective in stimulating the accumulation of components. At 150 ppm of micronutrients by biofertilizeres, en-hanced the production of citronellal, Chavicol. Although combination of H2Bo3- and Mn2+ at 300 ppm by mac-ronutrients in some of essential oils like neral, caryophyllene oxide and 14-hydroxy-Z-caryophyllene were more produced than 150 ppm combinations but most of essential oils significantly increased in 150 ppm concentration of micronutrients with macronutrients. Exo-Isocitral, Chavicol, 14-hydroxy-Z-Caryophyllene and Germacrene D, were upper extracted in 150 ppm concentration of micronutrients but in many combinations this essential oil was no extracted in little concentration.

Keywords: Lemon balm, nutrients, phytochemical

703Effects of macro/micronutrients on Lemon balm


1. Introduction

Lemon balm (Melissa officinalis L.) from Lamia-ceae family, a native of the northern Mediterranean region is cultivated as a medical herb. It is listed in a number of European Pharmacopoeia for its carmi-native, digestive, diaphoretic and stimulant activities (Sekeroglu et al. 2006). Lemon balm is one of the most important medicinal and aromatic plants, with antioxidant, antimicrobial, spasmolytic, astringent and specific sensorial properties. Essential oil of the plant, composed mainly of contains caryophyllene β, citronellal, geraniol, geranyl acetate, linalool, neral and ursolic acid, is responsible for some of these ef-fects (Bagdat and Cosge, 2006). Small amount of Mn2+ and H2Bo3- are essential for growth and quality of the crop because these micronutrients also control most of the physiological activities of the crop by in-terrupting the level of chlorophyll content in leaves which ultimately influence the photosynthetic activity of the plant. In micronutrients, Manganese is involved in many biochemical functions, primarily acting as an activator of enzymes such as dehydrogenases and de-carboxylases involved in respiration, amino acid and lignin synthesis, and hormone concentrations (Galavi et al. 2012). Boron made resistance of plasmalema and combination by other minerals and necessary for plants (Widom and Mihalkovic, 2008). Foliar fertil-izer is particularly useful technique which can be de-signed to meet plants specific needs for one or more micro or macronutrients especially trace minerals and enable to correct deficiencies, strengthen weak or damaged crops, speed growth and grow better and healthier plants. In a sense, biofertilizeres (beneficial microbial populations) are unique alternatives for the sustainable agriculture and safe production. Microbial inoculums not only increased the nutritional assimila-tion of plant (total N, P and K), but also improved soil properties, such as organic weight content, dissolved

organic carbon, dissolved organic nitrogen and total N in soil (Ge et al. 2014; Adak et al. 2014). Biofer-tilizeres have the social community of bacteria and other microorganisms that live in rhizosphere, colo-nize upon roots and as a consequence of this sym-biotic relationship; the host plant achieves a better growth. For instance, plant growth-promoting rhizo-bacteria (PGPR) are beneficial native soil bacteria that colonize on plant roots and result in increased plant growth (Yadegari et al. 2010). Micro and mac-ro elements play an important role in promoting the growth and production of plants. Micro elements participate in most of the enzymatic reaction and they also play a role indirectly through the synthesis of several growth regulators (Yadegari, 2015). N ad-dition increased the soil microbial biomass carbon (SMBC) and soil bacterial diversity. Moderate N (23, 46, 69 kg ha-1 yr-1) addition increased the soil bacterial diversity, whereas excess N (92 kg ha-1 yr-1) addition inhibited it. The SMBC and soil bacterial diversity were related to richness of plant functional groups (Sun et al. 2014). Fertilisation with micronu-trients can be effective in increasing the concentra-tion of micronutrient sat the soil-root interface. In addition, micronutrient-efficient crops and geno-types can increase an available nutrient fraction and hence increase micronutrient uptake. Physiological processes governing exudation and the soil-plant-microbe interactions in the rhizosphere is currently inadequate, especially in terms of spatial and tem-poral variability in root exudation as well as the fate and effectiveness of organic and inorganic com-pounds in increasing availability of soil micronutri-ents and undesirable trace elements. The interactions between microorganisms and plants at the soil root interface are particularly important as well obscure (Rengl, 2015; Viruel et al. 2014).

704 Yadegari


Although many studies performed about of macronu-trients effects on plants (Quezada et al. 2007; Wyllyam Do Vale et al. 2008; Khan et al. 2014; Raza et al. 2014; Sandana and Pinochet, 2014; Yañez-Mansilla et al. 2015), few studies examining micronutrients fertilizers and interaction of them by macronutrients in essential oil components on lemon balm have been conducted. Objectives of this study were to determine the effects of Manganese and Boron foliar applications with macro-nutrients (PO4

3- and NO3-) from biofertilizeres resourc-

es on some of plant growth parameters (leaf dry/fresh weight, root dry/fresh weight, stem/root length, number of stem) and essence components such as Neral, Chavi-col, Geraniol, Geranial, E-Caryophyllene, Caryophyl-lene oxide of Melissa officinalis L. at two seasons.

2. Materials and Methods

Seeds of Lemon balm were obtained from Iranian Seeds and Plant Improvement Institute. Seeds were planted in field condition. Table 1 shows physico-chemical properties of the soil. Treatments were micronutrient Mn and B levels and macronutrients (PO4

3- and NO3-) from biofertilizeres resources.

Field trials were established in 2013 and 2014 at Shahrekord (50°56/ E 32°18/ N) South Western Iran. Experiments were arranged in a randomized com-plete block design with a factorial layout and three replications. Topsoil of the experimental plot area was kept moist throughout the growing season when necessary.

Table 1. Some physical and chemical properties of soil for experiment (0 -30) cm.

Two foliar fertilizers Librel Mn and Unibor were ap-plied and all of them are mineral fertilizers. Librel Mn inclusive of 13% Mn chelated with EDTA and Unibor contains 15% B (obtained from The Chemical Company of England and Germany). Theses fertiliz-ers were sprayed at three concentrations (Mn1, Mn2, Mn3 were 0, 150 and 300 ppm of Mn, respectively and similar in B). Phosphate-solubilizing bacterium, Bacillus lichenifor-mis, in powder form applied at the level of 100 gr. ha-1 (P1 and P2 were used as symbols to show soil fortified with biofertilizer and without respectively). Nitroxin bio-fertilizer which its effective site is the group of nitrogen fixing bacteria belongs to Azospirillum and Azotobacter genera in liquid form applied at the level

of 4 l.ha-1 (N1 and N2 were used as symbols to show soil fortified with biofertilizer and without respectively). After soil test, the required nutrients were added to soil. At the beginning of the blooming stage, shoots of plants were harvested. Lemon balm shoots were cleaned with distillated water, and then were dried 3 days at 40 °C in oven. Micronutrients concentrations (Mn2+ and H2Bo3- in 0, 150 and 300 ppm) were employed since of plant-ing to beginning of flowering of plants in two steps by 2 week distance. The essential oils were analyzed by gas chromatography-mass spectrometry (GC/MS). Thermo Finnegan Trace 2000 GC/MS, made in the USA, was employed with a HP-5MS capillary column (30 m long and 0.25 mm wide, and a 0.25 μm of film thickness) at a 250 °C of injector chamber.



The initial column temperature was at 120 °C for 5 min then raised to 280 °C at the rate of 10 °C/min. Helium was used as a carrier gas at a rate of 35 ml/min. MS parameters were as follows: ionization en-ergy, 70 eV; ion source temperature, 200 °C; voltage, 3000 v; and mass range, 30 to 600. The compositions of the essential oil were identified by comparison of their retention indexes, retention times and mass spectra with those of authentic samples in Wiley li-brary (Adams, 2001). Amounts of essential oils components production as well as the chemical component of the essence were determined.All data were subjected to ANOVA using the sta-tistical computer package SAS ver.8 and treatment means separated using L.S.D multiple range test at P<0.05 level.

3. Results In various components, the best results made by con-sumption of 150 and 300 ppm of micronutrients with use of biofertilizeres. The most of essential oils in two seasons were Caryophyllene oxide, E-Caryophyllene, Geranial, Geraniol, Chavicol and Neral that affected by the treatments (Tables 3-6). Also, the micronu-trients application affected the caryophyllene oxide, E-Caryophyllene, Geranial, Geraniol, Chavicol and Neralpercentage. Similarly, the application of biofer-tilizeres increased the Caryophyllene oxide, E-Caryo-phyllene, Geranial, Geraniol and Chavicol of percent-age by 22%. The foliar application of H2Bo3- and Mn2+ affected the Caryophyllene oxide, E-Caryophyllene, Geranial, Geraniol, Chavicol and Neralof concentra-tion an average of 34% at 150 ppm application of mi-cronutrients compared with the control (Tables 3-4).

Source of variation Degree of freedom

Leaf dry weight

Leaf Fresh weight

Root dry weight

RootFreshweight

Stem length

Rootlength

Number of stem

Mean of Square Year(Y) 1 5.5* 10.1* 4.7* 7.5ns 24.1ns 21.6* 28.9*

R/Y 4 3.4* 6.22* 2.91* 5.1* 15.1* 13.36* 17.98*

Manganese(Mn) 2 7.5** 12.6** 3.67* 6.4* 18.9* 16.8* 22.65*

Boron (B) 2 7.7** 13.1** 3.6* 6.3* 19.1* 16.9* 22.71*

Nitrogen (N) 1 6.8** 15.91** 8.4** 8.2* 24.4* 21.7* 29.1*

Phosphorus (P) 1 7.8** 22.62** 10.1** 8.3* 24.2* 21.9* 29.2*

Mn×B 4 4.7** 12.1** 5.5** 5.12* 15.2* 13.35* 18.1*

Mn×N 2 5.7** 13.2** 6.6** 8.8** 41.1** 33.1** 41.1**

Mn×P 2 6.9** 12.2** 7.76** 9.3** 39.5** 33.7** 43.2**

B×N 2 6.2** 17.2** 8.91** 11.1** 51.6** 42.4** 51.5**

B×P 2 5.9** 13.4** 9.78** 12.4** 52.4** 45.5** 56.7**

N×P 1 5.9** 32.43** 12.3** 11.2** 45.5** 40.2** 29.05*

Mn×B×N 4 4.8* 14.5** 6.8** 8.5** 28.8** 28.1** 18.2*

Mn×B×P 4 4.8* 14.1* 8.81** 9.5** 44.2** 29.9** 18.3*

Mn×N×P 2 4.3* 7.8* 10.1** 8.1** 61.1** 41.7** 54.2**

B×N×P 2 4.12* 12.6** 12.2** 9.6** 59.2** 45.8** 56.1**

Mn×B×N×P 4 3.4* 16.2** 9.8** 4.95* 42.5** 32.5** 34.5**

T(Mn×B×N×P)×Y 35 2.1* 3.84* 1.8* 3.14* 9.3* 8.3* 11.1*

E 140 1.4 2.56 1.2 2.1 6.2 5.5 7.4 Coefficient of variation 2.74 5.5 1.5 3.4 8.9 5.9 5.3

Table 2a. Complex analysis of variance of essential oils components in essence in Lemon balm plants that are affected by micronutrients and biofertilizeres.

706 Yadegari


Table 2b. Complex analysis of variance of essential oils components in essence in Lemon balm plants that are affected by micronutrients and biofertilizeres.

Source of variation D.FNeral Chavicol Geraniol Geranial E-Caryophyllene Caryophyllene oxide

Year(Y) 1 0.015* 0.019* 0.0035* 0.0016* 0.0023* 0.0027*

R/Y 4 0.009* 0.012* 0.0021* 0.0008* 0.0014* 0.0017*

Manganese(Mn) 2 0.012* 0.015* 0.0027* 0.0013* 0.0018* 0.0021*

Boron (B) 2 0.013* 0.017* 0.0029* 0.0014* 0.0019* 0.0022*

Nitrogen (N) 1 0.016* 0.021* 0.0034* 0.0017* 0.0025* 0.0028*

Phosphorus (P) 1 0.017* 0.019* 0.0037* 0.0018* 0.0027* 0.0029*

Mn×B 4 0.011* 0.013* 0.0022* 0.0012* 0.0015* 0.0018*

Mn×N 2 0.024** 0.041** 0.0031* 0.0025** 0.0036** 0.0045**

Mn×P 2 0.028** 0.048** 0.0029* 0.0029** 0.0037** 0.0047**

B×N 2 0.029** 0.056** 0.006** 0.0031** 0.0041** 0.0051**

B×P 2 0.032** 0.066** 0.0072** 0.0034** 0.0045** 0.0054**

N×P 1 0.036** 0.043** 0.0081** 0.0037** 0.0024* 0.0027*

Mn×B×N 4 0.022** 0.012* 0.0024* 0.0024** 0.0035** 0.0039**

Mn×B×P 4 0.032** 0.044** 0.004** 0.0037** 0.0043** 0.0041**

Mn×N×P 2 0.061** 0.081** 0.0081** 0.0065* 0.0056** 0.0061**

B×N×P 2 0.055** 0.092** 0.0076** 0.006** 0.0045** 0.0055**

Mn×B×N×P 4 0.034** 0.051** 0.0032** 0.0044** 0.0061** 0.0044**

T(Mn×B×N×P)×Y 35 0.0061* 0.0075* 0.0014* 0.00062* 0.00091* 0.001*

E 140 0.004 0.005 0.0009 0.0004 0.0006 0.0007 Coefficient of variation 1.5 1.2 2.4 1.1 1.5 1.1

Biofertilizeres by H2Bo3- and Mn2+ applications affect-ed geranial, geraniol and geranyl acetate of stems and increased with the micro/macronutrients applications. There were statistical significant differences among the three rates of H2Bo3- and Mn2+ applications and application or/no application of biofertilizeres (Table 2). The amount of geranial per plant increased after the fertilizers application compared with the control by an average of (12%) (Tables 3, 5, 6). Linalool and neral concentrations were affected by biofertilizeres with H2Bo3- and Mn2+ applications and therefore increased in various range compared with the control (Tables 3-6). In generally the most of Caryophyllene oxide (8.4%), E-Caryophyllene (6.9%), Geranial (33.36%) and Chavicol (12.38%)

were made by treatments that had biofertilizeres with 150 ppm of Mn and B. In most of other subsidiary components contain of Linalool, exo-Isocitral, Citro-nellal, Z-Isoicitral, Rosefuran epoxide, E-Isoicitral, Piperitenone oxide, E-Caryophyllene, alpha-trans-Bergamotene, alpha-Humulene, Germacrene D, E-beta- Ionone, gamma-Cadinene, Humulene epoxide II, epi-alpha-Cadinol, 14-hydroxy-Z-Caryophyllene, Hexadecanoic acid and Abieta-8 (14),13 (15-diene); the treatments that had micronutrients with biofertil-izeres made most of components and in these treat-ments the treatment of Mn 150 ppm×B 150 ppm with use of biofertilizeres was the best. Biofertilizeres with micronutrients applications affected on chavicol per-centage of the crop (Tables 3-6).



Table 3a. Means of essential oil percentage measured in lemon balm plants that are affected by micronutrients (150 ppm) concentration and biofertilizeres (1st year).

Treatments 6-methyl-5-Hepten-2-1

Linalool exo-Isocitral Citronellal Z-Isoicitral Rosefuran epoxide

E-Isoicitral Methyl chavicol

Neral Chavicol E-Caryophyllene

Manganese(Mn) 1.43b 0.45ef 0.22b - 1.15bc 0.29c 1.73b 0.73d 24.09b - 7.54aBoron (B) 0.42e 0.44f 0.22b 0.37c 1.32b - 1.95ab - 19.05bc 7.33b 7.17a

Nitrogen (N) 1.36bc 0.49ef 0.22b 0.4c 1.27b 0.5a 1.8b 0.58d 14.14c 12.28a 7.37aPhosphorus (P) 1.06d 0.44f - 0.42bc 1.16bc 0.39c 1.65bc - 27.83a - 5.91b

Mn×B 1.26c 0.47ef 0.28a 0.5b 1.59a 0.53a 2.03a 2.82b 14.57c 12.65a 6.37abMn×N 1.31c 0.56de - 0.41c 1.08c 0.39b 1.56bc 2.01b 17.46c 6.16b 7.38aMn×P 1.19cd 0.39g 0.21b 0.35c 1.1c 0.4c 1.54c 4.1a 26.25a - 7.1aB×N 1.72a 0.25h 0.22b 0.56ab 1.28b 0.45bc 1.89b 4.31a 24.61b - 6.09bB×P 1.26c 0.48ef 0.23b 0.45bc 1.18bc 0.46bc 1.69bc 1.15c 25.59ab - 7.35aN×P 1.37bc 0.51e 0.2b 0.41c 1.29b 0.51a 1.7b 0.59d 14.4c 12.38a 7.16a

Mn×B×N 1.16d 0.54e - 0.62a 1.16bc 0.59a 1.6bc - 27.8a - 6.1abMn×B×P 1.6a 0.64d - 0.2d 1.32b 0.6a 2.05a - 19.8ab - 5.1cMn×N×P 1.46b 0.74c - 0.4c 1.36b 0.35c 1.75b - 19.9ab - 7.6aB×N×P 1.56ab 0.84b - 0.46bc 1.45ab 0.37c 1.05d - 18.8b - 6.6ab

Mn×B×N×P 1.32c 0.94a - 0.56ab 1.56a 0.59a 2.6a - 19.5ab - 6.8abControl 0.06f 0.44b - 0.49bc 1.23b 0.45bc 1.65bc - 20.3ab - 5.16c

Treatments alpha-trans-

Bergamotene Piperitone Methyl

citronellate Geraniol trans-

Piperitoneepoxide

Geranial Methyl nerolate

Nerylformate Methyl geranate

Piperitenone oxide

Geranyl acetate

Manganese(Mn) - 0.57b - 1.31b - 28.9b - - 0.47b - 4.9cBoron (B) - 0.64a 0.46a 1.44c - 20.81c 5.32a 6a 0.72a - 7.29a

Nitrogen (N) - 0.56b 0.4a 12.75a 1.37a 18.58c - - 0.49b - 5.44bPhosphorus (P) - 0.6ab 0.27bc 1.2c - 32.36a - - - - 4.85bc

Mn×B 0.5b 0.38c 0.32b 0.7c - 32.49a - - 0.46b 4.88a - Mn×N 1.12a 0.47c 0.27bc 1.05c - 27b - - - - 5.09bcMn×P 0.4b 0.7a - 1.15c - 31.39a - - 0.46b - 5.31bB×N 0.28b 0.39c - 0.81c - 19.19c 3.87b 5.3a 0.51b 1.11b 4.1cB×P - 0.47c 0.23c 0.77c - 30.82ab - - - 0.55c 6.07abN×P 0.39b 0.57b 0.4a 12.7a 1.4a 18.5c - 4.6b 0.44b - 3.42c

Mn×B×N 1.3a 0.6ab 0.27bc 1.42c - 32.36a - 1.6e 0.48b - 4.4cMn×B×P 1.9a 0.63ab 0.2bc 1.6c - 32.16a - 1.7e 0.49b - 3.5cMn×N×P 1.9a 0.45c 0.37b 1.8c - 32.36a - 2.6d 0.54b - 7.4aB×N×P 1.7a 0.56b 0.22c 7.9b - 32.6a - 2.6d 0.64a - 5.6b

Mn×B×N×P 1.9a 0.5b 0.23c 8.2b - 33.36a - 2.2d 0.4b - 3.4cControl 0.29b 0.66a 0.29b 9.2ab - 31.16a - 3.6c 0.14c - 3.4c

Table 3b. Means of essential oil percentage measured in lemon balm plants that are affected by micronutrients (150 ppm) concentration and biofertilizeres (1st year).

Treatments alpha-

Humulene Germacrene D E-beta-

Ionone gamma-Cadinene

Caryophyllene oxide

Humulene epoxide II

epic-alpha-

Cadinol

14-hydroxy-Z-Caryophyllene

Hexadecanoicacid

Abieta-8(14),13(15-

diene)

Total%

Manganese(Mn) 0.52bc - - - 12.46a 0.72a - 0.74a 0.29ab 2.59a 91.1 Boron (B) 0.47c - 0.24b - 9.79bc 0.49b - 0.6b - 1.75b 94.3

Nitrogen (N) - - 0.24b - 11.29ab - - 0.65ab - 2.19a 94.36 Phosphorus (P) 0.39c - 0.22b - 10.98b 0.56b - 0.61b - 2.11a 93.01

Mn×B 1.59a 0.93a 0.3ab 1.46a 6.37d - 0.4b 0.39c 0.24b - 94.49 Mn×N 0.71b 0.35b 0.42a 0.7b 12.62a 0.64ab 1.35a 0.64ab 0.26b 1.59b 92.6 Mn×P 1.29a 0.57b 0.3ab 1.12ab 8.13c 0.51b 0.23b 0.46c - 1.22b 95.89 B×N 0.88ab 0.4b 0.28b - 9.89bc 0.54b 0.29b 0.71a - 1.34b 91.24 B×P 0.55b - 0.23b - 11.48ab 0.61ab - 0.64ab - 2.04ab 94.3 N×P 1.5a - 0.3ab - 10.6b 0.2c 1.2a - 0.36a 1.3b 98.4

Mn×B×N 1.6a - 0.31ab - 8.4c 0.7a 1.3a - 0.3ab 1.64b 96.25 Mn×B×P 1.35a - 0.32ab - 12.3a 0.34c 1.02a - 0.3ab 2.34a 91.46 Mn×N×P 1.5a - 0.34ab - 8.6c 0.77a 1.12a - 0.32ab 2.64a 96.27 B×N×P 1.6a - 0.35ab - 6.6d 0.7a 1.42a - 0.4a 1.54b 95.56

Mn×B×N×P 1.65a - 0.38ab - 8.6c 0.57b 1.65a - 0.42a 2.64a 94.69 Control 0.65b - 0.3ab - 8.6c 0.5b 1.2a - 0.3ab 1.34b 91.41

Table 3c. Means of essential oil percentage measured in lemon balm plants that are affected by micronutrients (150 ppm) concentration and biofertilizeres (1st year).

708 Yadegari


Treatments alpha-trans-

Bergamotene 6-methyl-5-Hepten-

2-one

Linalool exo-Isocitral

Citronellal Z-Isoicitral

Rosefuranepoxide

E-Isoicitral

Methyl chavicol


Manganese(Mn) 1.77b 1.3c 0.43c 0.34a 0.43b 1.6a 0.52ab 1.82b 0.68d 19.1c 1.77cBoron (B) 1.23c 0.62e 0.46c 0.34a 0.39b 1.4ab - 1.86b - 21.8bc 4.32a

Nitrogen (N) - 1.4b 0.55c 0.33a - 1.21c 0.17d 1.7bc 0.6d 30.19ab - Phosphorus (P) - 1.6a 0.64bc - 0.44b 1.41ab 0.43b 1.85b - 35.6a -

Mn×B 0.5d 1.2cd 0.77ab 0.34a 0.51ab 1.59a 0.55ab 1.9b 2.8c 31.49a 1.75cMn×N 1.12c 1.31c 0.56c - 0.41b 1.08c 0.44b 1.54c 2.1c 27.3b - Mn×P 0.4d 1.19cd 0.39cd 0.21b 0.35d 1.1c 0.41b 1.56c 7.01a 32.3a - B×N 0.28d 1.72a 0.25d 0.22b 0.56a 1.28b 0.45b 1.89ab 4.31b 21.1bc 3.87aB×P - 1.26cd 0.48c 0.23b 0.45b 1.18c 0.46b 1.6bc 1.15c 29.8b - N×P 0.39d 1.3c 0.5c 0.2b 0.4b 1.29b 0.51ab 1.7bc 0.59d 21.5bc 2.7b

Mn×B×N 1.3c 1.12d 0.54c - 0.6a 1.16c 0.69a 1.64bc - 31.3ab 3.37abMn×B×P 1.9b 1.16d 0.5c - 0.62a 1.6a 0.59ab 1.6bc - 31.3ab 1.87cMn×N×P 1.8b 1.6a 0.6bc 0.2b 0.23d 1.39ab 0.66a 2.25a 0.55d 32.6a 3.5abB×N×P 1.8b 1.4b 0.74b - 0.48b 1.3b 0.35c 1.65bc 0.69d 28.6b 1.77c

Mn×B×N×P 2.1a 1.5ab 0.86a - 0.7a 1.6a 0.58ab 2.4a - 31.6ab 1.77cControl 1.9b 1.3c 0.7b - 0.45b 1.5a 0.39c 1.15d - 33.56a -

Table 4a. Means of essential oil percentage measured in lemon balm plants that are affected by micronutrients (150 ppm) concentration and biofertilizeres (2nd year).

Treatments Neral Chavicol Piperitone Methyl


Piperitoneepoxide


Piperitenone oxide

Geranyl acetate

E-Caryophyllene

Manganese(Mn) 14.3d 12.2a 0.59b 0.43a 2.75b 1.77a - 0.37c 1.77b 4.34bc 5.5bBoron (B) 18.5c 8.3b 0.68ab 0.49a 1.84c 1.41a 6.2a 0.7a - 7.2a 7.1a

Nitrogen (N) 24.9ab - 0.59b - 1.71c 1.47a - 0.21c 1.47b 5.4b 7.17aPhosphorus (P) 25.3a - 0.67ab 0.47a 1.23c - - - - 4.8bc 5.9b

Mn×B 15.5d 9.9b 0.42d 0.42a 0.77d - 1.7b 0.4b 4.8a - 6.3abMn×N 18.4c 6.6c 0.49d 0.47a 1.5c - - - - 5.2b 7.3aMn×P 22.25b - 0.71a - 1.85c - - 0.46b - 5.3b 7.11aB×N 24.61ab - 0.39d - 0.81d - 5.3a 0.51b 1.11b 4.1bc 6.09abB×P 25.59a - 0.47d 0.25a 0.77d - - - 0.55c 6.7a 7.35aN×P 14.2d 12.38a 0.57b 0.44a 2.7b 1.4a 5.6a 0.44b - 3.4c 7.16a

Mn×B×N 26.8a - 0.6ab 0.27b 1.4c - 2.6b 0.48b - 4.1bc 6.1abMn×B×P 25.8a - 0.61ab 0.27b 1.32c 1.2ab 1.9b 0.79a - 3.4c 5.1bMn×N×P 13.7d - 0.62ab 0.27b 1.26c 1.4a 2.5b 0.5b - 7.34a 7.7aB×N×P 15.6d 6.6c 0.35d 0.47a 1.28c 1.77a 2.4b 0.6a - 5.9b 6.9ab

Mn×B×N×P 18.4c 2.16d 0.54b 0.22b 2.9b 1.87a 2.9b 0.63a - 3.1c 6.9abControl 19.2c - 0.5b 0.23b 8.1a 0.9b 1.6b 0.72a - 3.6c 6.5ab

Table 4b. Means of essential oil percentage measured in lemon balm plants that are affected by micronutrients (150 ppm) concentration and biofertilizeres (2nd year).

Treatments alpha-



Caryophyllene oxide

Humulene epoxide II

epi-alpha-

Cadinol


Hexadecanoicacid

Abieta-8(14),13(15-

diene)

Total%

Manganese(Mn) 0.5d 1.37a 0.7a 1.77a 11.4a 0.7a 0.77b 0.44b 0.33b 2.5ab 94.26 Boron (B) 0.4d - 0.14c - 9.7bc 0.4b - 0.62a 0.37b 1.7bc 98.17

Nitrogen (N) - - 0.2c - 10.2b - - 0.61a - 2.1b 92.18 Phosphorus (P) 0.44d - 0.2c - 10.9b 0.5b - 0.6a - 3.1a 96.08

Mn×B 1.5b 0.9b 0.12c 1.4a 6.3d - 0.41c 0.44b 0.64a - 95.32 Mn×N 0.7d 0.45c 0.4b 0.72b 12.6a 0.14c 1.3ab 0.54ab 0.26b 1.5bc 94.43 Mn×P 1.3c 0.67bc 0.2c 1.1ab 8.1c 0.5b 0.2c 0.4b - 1.2c 96.27 B×N 0.88d 0.42c 0.28c - 9.8bc 0.5b 0.29c 0.7a - 1.4bc 93.12 B×P 0.55d - 0.23c - 11.4a 0.61ab - 0.64a - 2.4b 94.12 N×P 1.5b - 0.3b - 10.6b 0.22c 1.1ab - 0.36b 1.3bc 94.75

Mn×B×N 1.65ab - 0.41b - 6.4d 0.71a 1.2ab - 0.3b 1.6bc 96.34 Mn×B×P 1.45b - 0.42b - 8.3c 0.44b 1.2ab - 0.31b 2.3b 95.95 Mn×N×P 1.53b - 0.44b - 8.4c 0.74a 1.1ab - 0.32b 2.6ab 95.8 B×N×P 1.66ab - 0.45b - 6.3d 0.71a 1.4a - 0.43ab 2.5ab 96.1

Mn×B×N×P 1.66ab - 0.48b - 8.4c 0.5b 1.6a - 0.64a 2.6ab 99.15 Control 0.8a - 0.44b - 6.2d 0.6ab 1.4a - 0.41ab 1.5bc 93.65

Table 4c. Means of essential oil percentage measured in lemon balm plants that are affected by micronutrients (150 ppm) concentration and biofertilizeres (2nd year).



Table 5a. Means of essential oil percentage measured in lemon balm plants that are affected by micronutrients (300 ppm) concentration and biofertilizeres (1st year).

Treatments 6-methyl-5-Hepten-2-

one

Linalool exo-Isocitral Citronellal Z-Isoicitral Rosefuran epoxide


Neral Chavicol Piperitone

Manganese(Mn) 1.62a 0.46d 0.34a 0.39b 1.4a - 1.86b - 15.5cd 8.3a 0.8bBoron (B) 1.4b 0.54cd 0.34a - 1.21a 0.57b 1.75b 0.65d 24.5ab - 0.5c

Nitrogen (N) 1.4b 0.64b - 0.43b 1.43a 0.53b 1.8ab - 25.5a - 0.67bcPhosphorus (P) 1.24c 0.74b 0.34a 0.51b 1.53a 0.55b 1.95ab 2.58bc 15.5cd 9.9a 0.42c

Mn×B 1.41b 0.56cd - 0.41b 1.38a 0.44b 1.5bc 2.1c 18.4c 6.6b 0.4cMn×N 1.14c 0.39d 0.41a 0.33b 1.3a 0.45b 1.55b 7.51a 22.2b - 0.71bMn×P 1.14c 0.39d 0.24b 0.33b 1.3a 0.54b 1.54b 4.15b 19.5bc - 0.75bB×N 1.42b 0.45d 0.24b 0.53b 1.38a 0.45b 1.59b 4.31b 24.1ab - 0.39cB×P 1.46b 0.48d 0.23b 0.43b 1.38a 0.45b 1.69b 1.1c 25.59a - 0.4cN×P 1.3bc 0.54cd 0.2b 0.43b 1.29a 0.51b 1.7b 0.5d 14.1d 2.3c 0.5c

Mn×B×N 1.12c 0.54cd - 0.3b 1.13a 0.65b 1.64b - 26.8a - 0.65bcMn×B×P 1.46b 0.4d - 0.32b 1.3a 0.59b 1.56b - 25.8a - 0.61bcMn×N×P 1.6a 0.6c 0.24b 0.23b 1.39a 0.56b 2.5a 0.5d 13.5d - 0.6bcB×N×P 1.4b 0.44d - 1.03a 1.3a 0.35b 1.5bc 0.65d 15.6cd 6.6b 0.35c

Mn×B×N×P 1.2c 1.4a 0.2b 1.3a 1.2a 1.33a 2.1a - 18.1c 2.1c 0.5cControl 1.2c 0.81b - 0.43b 1.2a 0.3b 1.5b - 16.2c - 1.5a

Table 5b. Means of essential oil percentage measured in lemon balm plants that are affected by micronutrients (300 ppm) concentration and biofertilizeres (1st year).

Treatments Methyl


Piperitoneepoxide



Piperitenone oxide

Geranyl acetate

E-Caryophyllene

alpha-trans-Bergamotene

Manganese(Mn) 0.4b 1.8b 1.41ab 21.85bc 4.3a - 0.4ab - 5.4b 7.3a - Boron (B) - 1.7b 1.45ab 30.1ab - - - - 4.8bc 5.9b -

Nitrogen (N) 0.4b 1.23bc - 35.56a - - 0.4ab 4.8a - 6.3ab 0.5cPhosphorus (P) 0.42b 0.7c - 31.4a 1.7b - - - 5.9b 7.38a 1.2b

Mn×B 0.47b 1.5b - 27.35b - - 0.4ab - 5.31b 7.1a 0.34bMn×N - 1.8b - 32.3a - - 0.4ab - 5.3b 7.11a 0.43cMn×P - 1.15bc - 31.3a - 5.3a 0.5a 1.13b 4.13bc 6.9ab 0.8cB×N - 0.8c - 19.9c 3.8a - - 0.53c 6.73a 7.35a - B×P 0.3b 0.7c - 30.8ab - 5.6a 0.4ab - 3.4c 7.6a 0.3cN×P 0.44b 2.7b 1.4ab 21.5bc 2.7ab 5.2a 0.4ab - 3.43c 7.16a 0.39c

Mn×B×N 0.2b 1.4b - 31.35a 3.3a 2.1b 0.4ab - 4.1bc 6.13ab 1.33bMn×B×P 0.27b 1.3b 1.2b 31.3a 1.8b 1.2b 0.39b - 3.43c 5.13b 1.93aMn×N×P 0.2b 1.26b 1.45ab 32.65a 3.5a 2.2b 0.53a - 7.34a 7.7a 1.8aB×N×P 0.4b 1.2b 1.7a 28.6b 1.7b 2.2b 0.63a - 5.93b 6.39ab 1.8a

Mn×B×N×P 0.2b 2.91b 1.8a 32.6a 1.77b 2.2b 0.63a - 3.1c 6.93ab 2.13aControl 1.2a 6.5a 1.95a 31.5a - 1.6b 0.72a - 3.6c 6.5ab 1.9a

Treatments alpha-



Caryophyllene oxide

Humulene epoxide II

epi-alpha-

Cadinol


Hexadecanoicacid

Abieta-8(14),13(15-

diene)

Total%

Manganese(Mn) - - 0.23b - 11.29a - - 0.67a - 2.17ab 91.49 Boron (B) 0.3b - 0.22b - 10.98a 0.56b - 0.61a - 2.17ab 90.25

Nitrogen (N) 1.5a 0.93a 0.3ab 1.46a 6.37b - 0.47b 0.37b 0.27b - 93.26 Phosphorus (P) 0.71b 0.3c 0.4a 0.7b 5.62bc 0.64a 1.35a 0.64a 0.26b 1.57b 96.15

Mn×B 1.2a 0.5c 0.3ab 1.12a 8.13ab 0.57b 0.27b 0.47b - 1.22b 96.05 Mn×N 1.3b 0.6b 0.23b 1.1a 3.1c 0.57b 0.2b 0.4b - 1.27b 95.7 Mn×P 0.8b 0.4c 0.28b - 9.8ab 0.5b 0.27b 0.77a - 1.4b 95.31 B×N 0.5b - 0.23b - 11.4a 0.61ab - 0.67a - 2.4a 89.78 B×P 1.5a - 0.33ab - 10.6a 0.22c 1.1a - 0.3ab 1.37b 97.73 N×P 1.5a - 0.33ab - 10.6a 0.22c 1.1a - 0.37b 1.3b 84.11

Mn×B×N 1.6a - 0.41a - 6.4b 0.77a 1.2a - 0.3ab 1.6b 95.42 Mn×B×P 1.45a - 0.42a - 8.3ab 0.47b 1.27a - 0.31ab 2.37a 96.28 Mn×N×P 1.5a - 0.43a - 8.4ab 0.74a 1.1a - 0.37a 2.6a 95.49 B×N×P 1.66a - 0.45a - 6.3b 0.71a 1.4a - 0.43a 2.5a 93.22

Mn×B×N×P 1.6a - 0.43a - 8.4ab 0.57b 1.76a - 0.27b 2.67a 99.4 Control 1.8a - 0.44a - 6.2b 0.6ab 1.4a - 0.21a 1.5b 90.76

Table 5c. Means of essential oil percentage measured in lemon balm plants that are affected by micronutrients (300 ppm) concentration and biofertilizeres (1st year).

710 Yadegari


Table 6a. Means of essential oil percentage measured in lemon balm plants that are affected by micronutrients (300 ppm) concentration and biofertilizeres (2nd year).

Treatments 6-methyl-5-Hepten-

2-one

Linalool exo-Isocitral

Citronellal Z-Isoicitral Rosefuran epoxide


Neral Chavicol Piperitone

Manganese(Mn) 1.4c 0.32ab 0.22b - 1.5b 0.2b 1.7ab 0.75c 22.9ab - 0.57cBoron (B) 0.4d 0.33ab 0.22b 0.32c 1.2b 0.22b 1.9ab 0.6c 19.5b 7.33b 0.64c

Nitrogen (N) 1.2c 0.39ab 0.22b 0.41c 1.2b 0.5b 1.5b 0.48c 14.4bc 12.28a 0.56cPhosphorus (P) 1.6bc 0.4ab - 0.41c 1.1b 0.3b 1.6ab - 27.8a - 0.6c

Mn×B 1.2c 0.4ab 0.28b 0.51c 1.59b 0.3b 2.3a 2.82b 14.5bc 12.65a 0.38cMn×N 1.4c 0.56ab - 0.4c 1.8b 0.3b 1.5b 2.1b 17.4b 6.16b 0.47cMn×P 1.9b 0.39ab 0.21b 0.35c 1.1b 0.4b 1.5b 4.21a 26.5a - 0.7cB×N 1.7bc 0.25b 0.22b 0.5c 1.28b 0.45b 1.9ab 4.31a 23.61ab - 0.39cB×P 1.2c 0.48ab 0.23b 0.45c 1.18b 0.46b 1.6ab 0.15c 25.5ab - 0.47cN×P 1.9b 0.79a - 3.4a 5.1a 1.9a 1.45b - 1.42e - 2.3a

Mn×B×N 1.6bc 0.49ab 0.22b 0.4c 1.27b 0.5b 1.8ab 0.58c 10.14c 12.28a 0.56cMn×B×P 1.36c 0.49ab 0.21b 0.41c 1.2b 0.15b 1.6ab 0.5c 14.1bc 12.8a 0.5cMn×N×P 2.6a 0.48ab - 4.1a 3.1ab 1.3ab 1.65ab - 22.41ab - 1.4bB×N×P 1.9b 0.5ab - 3.4 a 5.1a 1.9a 1.45b 0.22c 12.42bc - 1.3b

Mn×B×N×P 1.5b 0.59ab 0.52a 3.34a 3.1ab 1.9a 1.9ab 0.62c 8.44d 10.1ab 0.56cControl 1.36c 0.49ab - 2.9a 2.9b 1.6ab 1.8ab 0.28c 8.14d - 0.56c

Table 6b. Means of essential oil percentage measured in lemon balm plants that are affected by micronutrients (300 ppm) concentration and biofertilizeres (2nd year).

Treatments Nerylformate Methyl

geranate Piperitenone

oxide Geranyl acetate

E-Caryophyllene alpha-trans-Bergamotene

alpha-Humulene

Germacrene D E-beta- Ionone

Manganese(Mn) 1.6b 0.4ab 2.2a 3.7b 7.2a 1.3b 1.52a 0.1b 0.33aBoron (B) 1.2b 0.3b 2.4a 3.4b 7.1a 1.3b 1.52a - 0.33a

Nitrogen (N) 2.1ab 0.5ab 2.2a 4.51ab 4.1b 1.3b 1.64a - 0.4aPhosphorus (P) 1.2b 0.3b 2.2a 3.4b 5.1ab 1.9a 1.45a 0.2b 0.4a

Mn×B 2.2ab 0.5ab - 7.3a 5.7ab 1.8a 1.5a - 0.33aMn×N 2.2ab 0.3b - 5.9ab 6.3a 1.8a 1.66a - 0.25aMn×P 2.2ab 0.3b - 3.15b 6.9a 2.13a 1.6a - 0.23abB×N 1.6b 0.2b - 3.65b 6.51a 1.9a 1.8a - 0.34aB×P - - 0.55b 6.7a 5.35ab - 0.55b - 0.23abN×P 2.6ab 0.42ab - 3.54b 5.6ab 0.3c 1.5a - 0.13b

Mn×B×N 3.2a 0.42ab - 3.43b 5.16ab 0.39c 1.5a 0.9a 0.13bMn×B×P 2.1ab 0.4ab - 4.1ab 5.1ab 1.33b 1.6a 0.2b 0.21abMn×N×P 1.2b 0.39ab - 3.43b 5.1ab 1.93a 1.45a 0.2b 0.22abB×N×P 2.2ab 0.53ab 2.3a 4.34ab 5.7ab 1.8a 1.5a 0.7a 0.23ab

Mn×B×N×P 2.2ab 0.63a 1.7a 4.93ab 5.3ab 1.8a 1.26a 0.8a 0.4aControl 2.2ab 0.63a 1.6ab 4.1b 5.4ab 1.63b 1.1a - 0.23ab

Table 6c. Means of essential oil percentage measured in lemon balm plants that are affected by micronutrients (300 ppm) concentration and biofertilizeres (2nd year).

Treatments gamma-

Cadinene Caryophyllene oxide Humulene

epoxide II epi-alpha-Cadinol


Hexadecanoic acid Abieta-8(14),13(15-diene)

Total %

Manganese(Mn) 1.2a 10.6a 0.12c 1.12a - 0.23a 1.3b 89.69 Boron (B) - 10.6a 0.2c 1.21a - 0.37a 1.32b 91.37

Nitrogen (N) - 9.4a 0.37b 1.22a - 0.32a 1.26b 95.49 Phosphorus (P) 0.2b 8.1a 0.27c 1.27a - 0.31a 1.37b 95.18

Mn×B - 3.4c 0.34b 1.12a - 0.37a 1.6ab 96.49 Mn×N - 6.3b 0.51a 1.4a - 0.43a 1.5ab 88.89 Mn×P - 8.4a 0.57a 1.76a - 0.27a 2.6a 99.82 B×N 1.2a 9.2a 0.6a 1.42a - 0.41a 2.5a 90.52 B×P - 9.48a 0.51a - 0.64a - 2.4a 89.93 N×P - 9.6a 0.32b 1.1a - 0.3a 1.3b 89.18

Mn×B×N - 8.6a 0.2c 1.12a - 0.37a 1.3b 97.66 Mn×B×P 1.1a 3.4c 0.47ab 1.2a - 0.3a 1.6ab 95.95 Mn×N×P 1.2a 6.3b 0.4ab 1.2a 0.2b 0.31a 2.3a 98.68 B×N×P 1.1a 6.1b 0.44ab - 0.5b 0.3a 2.2a 96.39

Mn×B×N×P 1.2a 6.3b 0.41ab 1.14a 0.7a 0.4a 1.25ab 98.99 Control 1.1a 6.4b 0.17c 1.06a - 0.27a 1.6ab 88.72

Numbers in each column that have same word, have same group. ns,*and** : Significant at P=0.05 and P=0.01 levels of probability, respectively.



4. Discussion

In the present study, the effect of micronutrients ap-plications with macronutrients (biofertilizeres) was determined on leaf dry/fresh weight, root dry/fresh weight, stem/root length, number of stem and essen-tial oils of Lemon balm. The most important charac-ters in this research was on essence components. In the soil application, the nutrients can be bound and therefore cannot be available for the plants. Also, since most nutrients are taken up with water, their uptake is restricted during water stress. The critical levels micronutrients were not determined for Lemon balm plants. In other hand, it was deduced dual inocu-lation with beneficial bacteria (Bacillus sp. N1-19NA, Enterobacter sp. N0-29PA, Pseudomonas sp.N1-55PA and Serratia sp. N0-10LB) are able to compen-sate the lack nutrient in soils (Martinez et al. 2015). Yield Enhancement in field trials was produced by us-ing a combination of Bacillus licheniformis, in pow-der form and Azospirillum and Azotobacter. Strains of bacteria have increased root and shoot elongation. The biofertilizer therefore may have a potential to de-crease the input cost of agricultural production, and be applied to the re-vegetation of low commercial value sites, such as metal tailings ponds (Yadegari et al. 2010). The effect of biofertilizeres on growth prop-erties, yield and quality property in medicinal plants was studied by other researchers and they showed same beneficial effects of biofertilizeres (Viruel et al. 2014). Patora et al. (2003) on Lemon balm and Yade-gari (2015) on borage, thyme and marigold showed beneficial application from micronutrients. The best treatment which was achieved maximum essence components of shoots was using biofertilizeres with micronutrients simultaneously. However, the L.S.D multiple range results revealed using biofertilizeres with micronutrients had the most influence on essence components of shoots and in various treatments, the

treat had 150 ppm of Mn and 150 ppm of B with use of biofertilizeres was the best (Tables 3-6). The most biological yield was obtained by biofertilizeres (Ya-degari et al. 2010). The enhanced phosphatase ac-tivity may help the plant to mobilize P and thereby increase the biomass production (Viruel et al. 2014). The significant effect that H2Bo3- and Mn2+ by biofer-tilizeres consumption had on essence and growth pa-rameters such as leaf dry/fresh weight, root dry/fresh weight, stem/root length, number of stem, indicates that micro/macronutrients play an important role in the yield of Lemon balm. Lemon balm needs No3-, Po4

3-, H2Bo3- and Mn2+ and it is possible that this re-quirement increases during growth, as the beginning of flowering process and the reproductive tissue have higher requirements for H2Bo3- and Mn2+ foliar appli-cations can have a direct effect on yield. In this study the response to micro/macronutrient sapplications can be higher. This is possible since the plants grew better with the macronutrients from biofertilizeres resource and micronutrients (H2Bo3- and Mn2+) application, de-veloped a bigger root system, and took up more nutri-ents. H2Bo3- and Mn2+ are immobile in plants and can’t be transported to developing organs. Therefore, foliar application can provide H2Bo3- and Mn2+ to the devel-oping organs that need it the most and, in this case, the plants do not have taken these micronutrients from the soil solution. However, the actual amount of H2Bo3- and Mn2+ that reaches the reproductive tissue can be small and depends on other factors such as H2Bo3- and Mn2+ soil and plant levels, and water stress that can restrict micronutrients movement and this is possibly one reason for finding a significant increase in yield because of the micronutrients application. Micro/mac-ronutrients application affected exo-Isocitral, Chavi-col, 14-hydroxy-Z-Caryophyllene and Germacrene D as the plants in the control treatment and increased with biofertilizeres with micronutrients applications and no detected in control plants (Tables 3-6).

712 Yadegari


In same research, the effect of farm yard manure reported significant on neral, (28.43%), geranial (39.86%) and geranyl acetate (8.67%) compared to other treatments (Harshavardhan et al. 2007).When the nitrogen, phosphorus, boron and manganese level is too low to sustain plant growth, the plants be-come shorter and the total biomass is lower compared with the plants with sufficient micronutrients (Mengel et al. 2001; Habib, 2012). This effect can be explained as micro/macronutrients affecting the carbohydrate transport, and this can affect the yield components in many plants. Since micro/macronutrients affects the meristems, the increase in the availability of micro/macronutrients can increase the number of stems per plant as they grow better.Galavi et al. (2012) on saf-flower, Yarnia et al. (2012) on Purple coneflower, Younis et al. (2013) on Rosa hybrida, Marquez et al. (2015) on cowpea, Yadegari (2015) on borage, thyme and marigold, found that spraying micronutrients in-creased essential oils in plants. The essential oil yield increased with H2Bo3- and Mn2+ applications with mac-ronutrients, because there was a significant increase in dry matter yield. However, more research is needed to explore these tools for Lemon balm breeding and for better Lemon balm management. There are still many unanswered questions about how biofertilizeres with H2Bo3- and Mn2+ act in increasing essential oils for Lemon balm. One possibility is that the applied micronutrients can affect dry matter accumulation and by macronutrients consumption increase dry matter yield. Macro/micronutrients can increase the number of stems per plant and slowdown leaf senescence (Ya-degari, 2015). The obtained results are in conformity with those of Sari and Ceylan (2002) on lemon balm and Yadegari (2015) on borage, thyme and marigold. The main components of the oil were Caryophyllene oxide, E-Caryophyllene, Geranial, Geraniol, Chavicol and Neral for all treatments. Many researchers have reported that the main components of lemon balm are

Caryophyllene oxide, E-Caryophyllene, Geranial, Ge-raniol, Chavicol and Neral (Manukyan, 2011; Patora et al. 2003; Sari and Ceylan, 2002). However, there were significant differences among the rates of those reported components. Neral and geranial rates in the oil were reported respectively as 15% and 14.5% by Hefendehl (1970), 19.6-36.1% and 25.3-47.5% by Tittel et al. (1982), 19.5% and 31.6% by Werker et al. (1985), 7.195% and 30-40% and 50-60% by Ceylan et al. (1994). Although many studies discussed above reported that neral and geranial were the main compo-nents of the oil, Kirimer et al. (1995) found that the main component of the lemon balm oil they studied in some of studies citronellal, linalool and geranial as major chemical compositions of the essential oil of the lemon balm (Adinee et al. 2008; Bagdat and Cosge, 2006; Mrlianova et al. 2001; Sorensen, 2000). All the significant differences for the components of the oil among the papers discussed above may be due to the use of different genetic material and/or different environmental conditions.

5. Conclusions

This study showed that the applications macronutri-ents (PO4

3- and NO3-) from biofertilizeres with micro-nutrients (H2Bo3- and Mn2+) had significant effects on growth parameters such as leaf/root dry/fresh weight, stem/root length, number of stem and essential oils of Lemon balm such as trans-Piperitone epoxide per-centage. The main components of the oil were Caryo-phyllene oxide, E-Caryophyllene, Geranial, Geraniol, Chavicol and Neral for all treatments. This indicates that application of micronutrients and soil consump-tion of macronutrients from biofertilizeres have sig-nificant effects on the composition of the essential oil. Results obtained from this study showed that the essential oils composition of lemon balm were signifi-cantly affected by application of micronutrients and



biofertilizeres. The highest amount of essential oil compositions such as Linalool, Z-Isoicitral, Rosefu-ran epoxide, E-Isoicitral, alpha-trans-Bergamotene, alpha-Humulene, epi-alpha-Cadinol, Hexadecanoic acid and Abieta-8 (14),13 (15-diene) obtained from combination of treatments of micronutrients and bio-fertilizeres application in two cultivation seasons. This study provides some useful information about the effects of application of PO4

3-, NO3-, H2Bo3- and Mn2+ on Lemon balm production and in that way in-creases our knowledge about the effect of micro/mac-ronutrients on crop production.

Acknowledgement

This work was supported by the Islamic Azad Uni-versity Branch of Shahrekord, Iran. We are very grateful to Mrs. P. Karimi from Education Center of Shahrekord for helpful advice for secondary reviewer.

References

Adams, R.P. 2001. Identification of Essential Oil Components by Gas Chromatography/ Mass Spectroscopy. Allured publishing Corp. Carol Stream. USA.

Adak, T., Singha, A., Kumar, K., Shukla, S.K., Singh, A., Kumar Singh, V. 2014. Soil organic carbon, dehydrogenase activity, nutrient availability and leaf nutrient content as affected by organic and in-organic source of nutrient in mango orchard soil. Journal of Soil Science and Plant Nutrition. 14(2): 394-406.

Adinee, J., Piri, K.H.,Karami, O. 2008. Essential Oil Component in Flower of Lemon Balm (Melissa officinalis L.). American Journal of Biochemistry and Biotechnology. 4(3): 277-278.

Bagdat, R.B.,Cosge, B. 2006. The essential oil of Lemon balm (Melissa officinalis L.), its compo-nents and using fields. Journal of Faculty of Agri-culture OMU. 21(1): 116-121.

Ceylan, A., Bayram, E.,Ozay, N. 1994. Melissa offi-cinalis L. (Oguloto) in agronomikveTeknolojik-Ozellikleri Uzerinde Arastirmalar. Doga, Turkish Journal of Agricultural and Forestry. 18, 125-130.

Galavi, M., Ramroudi, M.,Tavassoli, A. 2012. Effect of micronutrients foliar application on yield and seed oil content of safflower (Carthamus tincto-rius). African Journal of Agricultural Research. 7(3): 482-486.

Ge, P., Da, L.G., Wang, W.B., Xu, X.N. 2014. Season-al dynamics of dissolved organic carbon, nitrogen and other nutrients in soil of Pinus massoniana stands after pine wilt disease disturbance. Journal of Soil Science and Plant Nutrition. 14(1): 75-87.

Habib, M. 2012. Effect of supplementary nutrition with Fe, Zn chelates and urea on wheat quality and quantity. African Journal of Biotechnology. 11 (11): 2661-2665.

Harshavardhan, P.G., Vasundhara, M., Sreenivasappa, K.N. 2007. Influence of spacing and integrated nutrient management on yield and quality of es-sential oil in lemon balm (Melissa officinalis L). BIOMED. 2(3): 288-293.

Hefendehl, F.W. 1970. Zusammensetzung des atherischen Ols von Melissa officinalis L. und sekundareVeranderungen der Olkomposition. Arch. Pharm. 303, 345-357.

Khan, I., Zaman, M., Khan, M.J., Iqbal, M., Babar, M.N. 2014. How to improve yield and quality of potatoes: effects of two rates of urea N, ure-ase inhibitor and Cytozyme nutritional program. Journal of Soil Science and Plant Nutrition. 14 (2): 268-276. http://dx.doi.org/10.4067/S0718-95162014005000022.

714 Yadegari


Kirimer, N., Baser, K.H.C.,Tumen, G. 1995. Carvacrol-rich plants in Turkey. Khim. Prir. Soedin. 4: 49-54.

Manukyan, A. 2011. Effect of growing factors on pro-ductivity and quality of Lemon catmint, Lemon balm and Sage under soilless greenhouse produc-tion: I. Drought stress. Medicinal and Aromatic Plant Science and Biotechnology. 5(2): 119-125.

Martínez, O.A., Crowley, D.E., Mora, M.L., Jorquera, M.A. 2015. Short-term study shows that phytate-mineralizing rhizobacteria inoculation affects the biomass, phosphorus (P) uptake and rhizosphere properties of cereal plants. Journal of Soil Science and Plant Nutrition. 15(1): 153-166.

Márquez-Quiroz, C., De-la-Cruz-Lázaro, E., Osorio-Osorio, R., Sánchez-Chávez, E. 2015. Bioforti-fication of cowpea beans with iron: iron´s influ-enceon mineral content and yield. Journal of Soil Science and Plant Nutrition. 15 (4): 839-847.

Mengel, K., Kirkby, E.A., Kosegarten, H.,Appel, T. 2001. Principles of Plant Nutrition. Kluwer Aca-demic Publishers, London.

Mrlianova, M., Tekelova, D.,Grancai, D. 2001. Com-parison of the quality of Melissa officinalis L. cultivar Citra with Mellissas of European origin. Pharmacospsychiatry. 34: 20-21.

Patora, J., Majda, T., Gora, J.,Klimek, B. 2003. Vari-ability in the content and composition of essential oil from Lemon balm (Melissa officinalis L.) cul-tivated in Poland. Acta Poloniae Pharmaccutica. 60(5): 395-400.

Quezada, C., Vidal, I., Lemus, L., Sanchez, H. 2007. Effect of nitrogen fertilization on yield and fruit quality of raspberries (Rubusidaeus L.) under two fertigation programs. R.C. SueloNutr. Veg. 7(3): 1-15. ISSN 0718-2791. http://dx.doi.org/10.4067/S0718-27912007000300001.

Raza, M.A.S., Saleem, M.F., Shah, G.M., Khan, I.H., Raza, A. 2014. Exogenous application of gly-cinebetaine and potassium for improving water

relations and grain yield of wheat under drought. Journal of Soil Science and Plant Nutrition. 14(2): 348-364. http://dx.doi.org/10.4067/S0718-95162014005000028.

Rengel, Z. 2015. Availability of Mn, Zn and Fe in the rhizosphere. Journal of Soil Science and Plant Nutrition. 15 (2): 397-409. Sandana, P., Pino-chet, D. 2014. Grain yield and phosphorus use efficiency of wheat and pea in a high yielding environment. J. Soil Sci. Plant Nutr.14 (4): 973-986. ISSN 0718-9516. http://dx.doi.org/10.4067/S0718-95162014005000076.

Sari, A.O.,Ceylan, A. 2002. Yield Characteristics and Essential Oil Composition of Lemon balm (Me-lissa officinalis L.) Grown in the Aegean Region of Turkey. Turkish Journal of Agriculture. 26: 217-224.

Sekeroglu, N., Alpaslan, D., Kirpik, M. 2006. Es-sential oil contents and ethno pharmacological characteristics of some species and herbal drugs traded in turkey. International Journal of Pharma-cology. 2(2): 256-261.

Sorensen, J.M. 2000. Melissa officinalis, Essential oil-authenticity, production and pharmacological activity. International Journal Aromatherapy. 10: 1-8.

Sun, S., Xing, F., Zhao, H., Gao, Y., Bai1, Z., Dong, Y. 2014. Response of bacterial community to simu-lated nitrogen deposition in soils and a unique re-lationship between plant species and soil bacteria in the Songnen grassland in Northeastern China. Journal of Soil Science and Plant Nutrition. 14(3): 565-580.

Tittel, G., Wagner, H., Bos, R. 1982. Chemical com-position of the essential oil from Melissa. Planta Medica. 46, 91-98.

Viruel, E., Erazzú, L.E., Martínez Calsina, L., Fer-rero, M.A., Lucca, M.E.,Siñeriz, F.E. 2014. In-oculation of maize with phosphate solubilizing



bacteria:effect on plant growth and yield. Journal of Soil Science and Plant Nutrition. 14 (4): 819-831.

Yadegari, M., Asadi Rahmani, H., Noormohammadi, G. 2010. Plant growth promoting Rhizobacteria increase growth, yield and nitrogen fixation in Phaseolus vulgaris. Journal of Plant Nutrition. 33, 1733–1743.

Yadegari, M. 2015. Foliar application of micronutri-ents on essential oils of borago, thyme and mari-gold. Journal of Soil Science and Plant Nutrition. 15, 949-964.

Yañez-Mansilla, E., Cartes, P., Reyes-Díaz, M., Ri-bera-Fonseca, A., Rengel, Z., Alberdi, M. 2015. Leaf nitrogen thresholds ensuring high antioxi-dant features of Vaccinium corymbosum culti-vars. J. Soil Sci. Plant Nutr. 15, 3, 574-586.

Yarnia, M., Farzanian, M., Aliasgharzad, N. 2012. Ef-fects of microelement fertilizers and phosphate biological fertilizer on some morphological traits of Purple coneflower in water stress condition. African Journal of Microbiology Research. 6, 4825-4832.

Younis, A., Riaz, A., Sajid, S.,Nadeem, M. 2013. Fo-liar application of macro- and micronutrients on the yield and quality of Rosa hybridacvs. Cardinal and Whisky Mac. African Journal of Biotechnol-ogy. 12(7): 702-708.

Werker, E., Ravid, U., Putievsky, E. 1985. Structure of glandular hairs and identification of the main components of their secreted material in some species of the Labiatae. Israel Journal of Botany. 34, 31-45.

Widom, M., Mihalkovic, M. 2008. Symmetry-broken crystal structure of elemental boron at low tem-perature. Physiology Review B. 77 (6): 24-34.

Wyllyam Do Vale, D., De Mello Prado, R., Na-tale, W., Ursulino Alves, A. 2008. Nutritional response of rootstock of lemon ‘citrumelo’ to nitrogen, phosphorus and potassium applica-tion. R.C. Suelo Nutr. Veg. 8(3): 40-48. ISSN 0718-2791. http://dx.doi.org/10.4067/S0718-27912008000300004.

effect of micronutrients foliar application and ... · they also play a role indirectly through the...

Documents